A. Field of the Invention
The present invention relates generally to the field of cell culture. In particular, the invention relates to devices and methods that allow for the growth maintenance of cell cultures at high cell density. Some aspects of the invention involve a perfusable culture device designed so that a constant culture media level is maintained. Other aspects of the invention involve the use of the devices of the invention for time-lapse cinemicrography.
B. Background of the Related Arts
Traditional tissue culture procedures involve growing or maintaining cells in liquid or on solid media in culture flasks. One of the limitations of this type of system has been the fact that it is difficult to grow high-density tissue cultures in such a system.
Cells maintained in culture systems frequently have defined and stringent nutrition needs. In order to meet these needs, it has been necessary to maintain a relatively high media-to-cell ratio. Otherwise, the cells rapidly deplete the media of nutritive components and fill the media with metabolic waste. Further, CO.sub.2 gas bubbles build up and obscure the growing cells when one attempts to grow the cells to high density. This leads to difficulty in recording the growth of cells in culture via time lapse photography.
In order to forestall these problems, traditional cell culture has involved growing cells until they are just confluent, then trypsinizing the cells to remove them from the culture vessel and placing a portion of the cells in a new vessel with fresh media. Allowing cells to grow into a higher density is difficult with flasks because once the media becomes low in nutritive value, the cells either die or grow in aberrant manners. There is a current need in the field of cell growth research, e.g. in clinical organogenesis, for a system which allows for the growth of dense tissue cells which are multiple cell-layers thick. One favorable characteristic of such a system could be that it would maintain a constant level of culture media within the device. Unfortunately, there has been no system which readily permits such a constant culture media level.
Another form of culture used by researchers is organ culture, where whole or sliced animal organs are grown in culture media. Organ culture confronts the same problems as cell culture in that the need for nutrients requires a high media-to-cell ratio. Further, organ culture also confronts problems because it is difficult to diffuse nutrients into the center of thicker masses of organ tissue.
One of the current frontiers in the field of cell growth is the study of how cells live and grow in vivo. In vivo, cells are in contact with each other, many of the cells are quiescent, and cells undergo morphogenesis into various tissue types. Each of these states of cell growth and development has been difficult to observe in traditional tissue culture systems. The known culturing systems have not allowed cells to be grown until they reach a quiescent state where morphogenesis can occur. This is because of the constant need to cycle cell media and reduce the number of cells. Further, it has been difficult to study and perform organogenesis, a subset of morphogenesis. In organogenesis, a population of cells morphologically differentiates such that an organ is formed. While there has been some limited success at growing very thin, often single cell thick, layers of skin for medical use. The growth of thicker tissues and organs for clinical use has proven difficult. A large part of this difficulty has been the inability to constantly supply nutrients to the growing cells in a system where the media-to-cell ratio is low.
Time lapse cinemicrography devices provide a valuable tool for the study of cell growth and differentiation. While systems for such studies have been known, the data obtained have been limited since only low density cell cultures have been grown for long periods of time. The prior art tissue culture chambers are not suited for growing high density tissue cultures for periods long enough to allow for tissue morphogenesis to occur.
Prior time lapse cinemicrography studies have usually involved observation of individual cells at high magnification. In such studies, cells have been maintained at very low density since many microscopic features of cells are obscured under confluent conditions. For such studies, the Rose (Rose 1954) and Sykes-Moore chambers (Sykes, et al., 1959) have proven to be quite satisfactory. The present inventors attempted to use the Sykes-Moore chamber for time-lapse studies of cells maintained at very high cell density and found that several problems occurred even when the Sykes-Moore chamber was perfused several times a day with medium delivery by a peristaltic pump. The chief problems encountered were the production of CO.sub.2 gas bubbles which interrupted the optical path and resulted in constant defocusing of the system due to the deformation of the coverslip walls of the chamber.
Time-lapse cinemicrography studies of cells maintained at very high cell density in Sykes-Moore chambers even with a medium perfusion system, gave unsatisfactory results for several reasons. First, CO.sub.2 produced by cells led to the formation of gas bubbles which often obstructed the optical path. Second, the specimen regularly went out of focus due to gas pressure build up which caused the glass coverslips to warp, and, in some cases, break. Third, after several days cells underwent degenerative changes apparently due to lack of oxygen.
To observe morphological changes in cells maintained at high cell density studies were initiated with Sykes-Moore chambers. In such studies, MDCK cells were planted in Sykes-Moore chambers that had been completely filled with medium and connected to a peristaltic pump which perfused the culture. Gas bubbles formed once cells had become confluent and that the microscope appeared to continually go out of focus in spite of several attempts to bolster the stage lock mechanism.
By sealing all orifices with silicone rubber glue, it was determined that the formation of gas bubbles was not due to a leak in the peristaltic pump tubing or chamber itself. The present inventors concluded that the gas bubbles were due to CO.sub.2 produced by the cells. The constant problem of the microscope going out of focus was determined to be due to CO.sub.2 pressure deforming the glass coverslips that make up the top and bottom of the Sykes-Moore chamber. Indeed, on several occasions, the glass coverslips of the Sykes-Moore chamber split due to gas pressure build-up.
These problems caused the Sykes-Moore chamber to be of use for only about one day when observing cells at moderately high density. This is too short to allow for dense cultures to grow and for meaningful time-lapse micrography.
The failure of existing technology forced the present inventors to develop a new culture apparatus that would allow cells to grow to high cell density and allow long-term time-lapse cinemicrography.
Initially, Applicants attempted to maintain a constant media level in profusion chambers by having a single culture media inlet and a single culture media outlet, both positioned below the desired fluid level. The idea was that if culture media could be pumped into the device at exactly the same rate it was being pumped out of the device, a constant fluid level would be maintained. While theoretically workable, this configuration proved to be very difficult to place in practice. It was found to be almost impossible to obtain exactly equal inflow and outflow of the media. Therefore, over a period of time, the culture chamber would either fill with media or be drained below the desired level. This can cause at least two problems: (1) cells die in an empty chamber, and (2) even if the chamber were not to become completely empty or full, a constantly shifting media level will obscure the focus necessary for time lapse micrography.
Confronted with these problems, the present inventors created a simple, dependable, and inexpensive system for maintaining high density tissue cultures for a long period of time.